Artist’s rendition of protein ribbons

Protein purification is an essential component of protein research. The study of protein function, structure, and interactions heavily relies on the purity and quality of the isolated protein of interest. Here we present to you a five-step workflow that will help you in your quest. Learn about methods and technologies for protein expression, protein extraction and preservation, protein purification, protein clean up, and protein quantitation and detection.


Follow these 5 steps to obtain optimal protein sample conditions

Choosing the right expression system for your production needs

Researchers have many methodology choices when it comes to producing recombinant proteins for early-stage discovery research through large-scale production of biotherapeutic drugs, vaccine development, and structural studies. It is imperative to use the right protein expression system for the target protein and application of interest. We offer a wide selection of superior mammalian, insect, bacterial, and yeast protein expression systems to suit your research needs.

Our Gibco Expi Transient Expression Systems, available in mammalian (CHO-S, 293F cells) and insect (Sf9 cells) expression formats, are completely optimized systems that enable rapid, high-yield production of proteins.

Icon-based chart showing the steps of protein expression from cells to purified protein
Bar charts of titer vs expression system for 3 different proteins

Recombinant protein titers in ExpiCHO and other transient CHO expression systems. Expression levels of human IgG, rabbit IgG and erythropoietin in ExpiCHO and other transient CHO expression systems are shown. ExpiCHO titers range from 25–160 times those of other transient CHO expression systems.

Product highlight Description
ExpiCHO Expression System The Gibco ExpiCHO Expression System is a completely optimized system consisting of ExpiCHO-S(TM) cells that have been adapted to high-density, serum-free suspension culture in ExpiCHO(TM) Expression Medium, along with specially designed transfection reagents and enhancers, that provide the highest yields possible in a transient system (up to 3g/L). That means you can start your research work in CHO cells and stay in CHO cells through discovery.
Expi293 Expression System The Gibco Expi293 Expression System is a completely optimized system consisting of Expi293F(TM) cells that have been adapted to high-density, serum-free suspension culture in Expi293(TM) Expression Medium, along with specially designed transfection reagents and enhancers, that enable recombinant 293-derived recombinant protein expression in just five to seven days, with a 2-10-fold increase in protein yields compared to previous generation transient expression systems
ExpiSf Expression System The ExpiSf Expression System is the first-ever chemically defined baculovirus-insect cell protein expression system, delivering superior yields (3x more protein) and consistent performance run after run using a fast, streamlined workflow.

Tips

Make sure to check for the following things when getting started with an Expi transient expression system:

  • Check your DNA: 260:280 ratio, 260:230 ratio, and the DNA concentration prior to transfection
  • Check your reagent storage conditions: 4-8°C and protected from light
  • Check your incubator: ensure display temperature, CO2%, and humidity are accurate and at 37°C, 8%, and 80% respectively
  • Check your cells: ensure doubling time, viability and cell diameter are all ideal and ready for transfection
  • Check your volumes: make sure you have the culture volume: flask size ratio of 1:3.6 (1:3 – 1:4 is ok)
  • Check your speed: decrease speed with increased volume; increase speed with decreased volume
Discover more about protein expression


Protein expression products

Product selection guides Products
Transient protein expression systems Expi293
ExpiCHO
ExpiSf Expression Systems
Mammalian protein expression Expi293
ExpiCHO Expression Systems
Insect protein expression ExpiSf Expression System
Bacterial protein expression Champion pET Expression System
Yeast protein expression PichiaPink Yeast Expression System
Structural Biology and Membrane Protein Expression Expi293 GnTI-
Expi293 Inducible
Expi293 Inducible GnTI- Expression Systems
Expi293 Met(-) Protein Labeling Kit
Protein expression selection guide Protein Expression Systems Selection Guide
Protein expression services Gene-to-Protein
Baculovirus Expression
Mammalian Expression services

Extract and stabilize your target protein from the sample

Protein extraction techniques vary depending on the source of the starting material, the location of the protein of interest within the cell, and the downstream application. Other important considerations include the preservation of protein activity and function as well as the reduction of background effects.

Protein extraction

Tissue and cell lysis

Historically, mechanical disruption has been used to lyse cells and tissues; our gentle, detergent-based solutions have been developed to efficiently lyse cells and enable the separation of subcellular structures without requiring physical disruption, providing high yields of active proteins.

Protein extraction product features:

  • Optimized—formulations maximize protein yield and preserve protein activity
  • Efficient—only produces minimal cross-contamination between subcellular fractions
  • Compatible—extracts can be used directly in most downstream applications
  • Gentle—eliminates the need for mechanical cell disruption for most sample types

High protein yield from a variety of mammalian cell types and cellular compartments

Western blot results to identify 7 different cellular proteins from 4 cell line lysates and 2 primary cell lysates

Protein extraction efficiency from major cellular compartments using M-PER Mammalian Protein Extraction Reagent. Lysates from established cell lines and primary cultures were prepared using M-PER reagent and extraction efficiency from the various cellular compartments evaluated. For each target protein, 10 µg of lysate was loaded for and electrophoresed by SDS PAGE, transferred to nitrocellose membrane and detected by western blot using SuperSignal West Pico PLUS Chemiluminescent Substrate.

Bar chart of protein yield per 10e6 cells vs cell line for 4 cell line lysates and 2 primary cell lysates

Protein yield from various cell types using M-PER Mammalian Protein Extraction Reagent. Cells were harvested at 85% confluency, washed twice and collected in ice-cold PBS and counted. For each cell type, 1 x 106 cells were pelleted by centrifugation at 2,000 x g for 5 minutes and lysed in 1 mL M-PER Reagent for 5 minutes. The cell lysates were clarified by centrifugation at 14,000 x g for 10 minutes and the supernatant was collected and the protein concentration (µg/million cells) was determined using the Pierce BCA Protein Assay.

Efficient and selective enrichment of membrane proteins

Improved protein yield using the Thermo Scientific Mem-PER Plus Membrane Protein Extraction Kit. Membrane proteins were isolated from mouse liver tissue and HeLa cells using Thermo Scientific Mem-PER Plus Membrane Protein Extraction Kit and three other commercial extraction kits. Protein yields (μg) for membrane, cytosolic, and total fractions were determined using the Thermo Scientific Pierce BCA Protein Assay Kit.

Efficient protein extraction from a variety of tissue types

Efficient extraction from bacterial cells

Protein yield comparison of two bacterial cell lysis reagents. E. coli ER2566/pLATE51-Klenow, ER2566/pGST-CC-StpB, and ER2566/pGS-Syk cell pellets (0.5 g), were resuspended in 2.5 mL aliquots of Thermo Scientific B-PER Complete Bacterial Protein Extraction Reagent or EMD Chemicals BugBuster Master Mix with gentle vortexing for 15 minutes at room temperature. Insoluble cell debris was removed by centrifugation at 16,000 x g for 20 minutes at 4°C. Protein yields (concentrations) for soluble fractions were determined using the Pierce BCA Protein Assay Kit.

Sample type Goal Product highlight
Primary or cultured mammalian cells or tissues Total protein extraction M-PER reagent
T-PER reagent
N-PER reagent
RIPA Lysis and Extraction Buffer
IP Lysis Buffer
Cultured mammalian cells or tissues Subcellular fractionation or organelle isolation NE-PER reagent
Subcellular Fractionation Kits
Mitochondria Isolation Kits
GPCR Extraction and Stabilization Reagent
Cell Surface Protein Isolation Kit
Syn-PER Reagent
Lysosome Enrichment Kit
Bacterial cells Total protein extraction B-PER reagent  
Yeast cells Total protein extraction Y-PER reagent  
Insect cells (baculovirus) Total protein extraction I-PER reagent  
Plant tissue (leaf, stem, root, flower) Total protein extraction Plan Total Protein Extraction Kit  

Detergent solutions

Detergents are frequently used in cell lysis reagent formulation and other protein research methods. Thermo Scientific Surfact-Amps Detergent Solutions are highly purified, precisely diluted (10%) formulations that are ideal for applications or assays that are sensitive to contaminants present in unpurified detergents.

Protein detergent product features:

  • Accurate—precise 10% detergent solution in ultrapure water
  • Easy-to-use—solution is simple to dispense and dilute
  • Exceptionally pure—less than 1.0 μq/mL peroxides and carbonyls
  • Stable—packaged under inert nitrogen gas in glass ampules or HDPE bottles
Graphic of simplified detergent molecule showing hydrophobic tail and hydrophilic head

Generic structure of a detergent molecule.

Protein stabilization

Cell lysis disrupts cell membranes and organelles, resulting in unregulated enzymatic activity that can reduce protein yield and function. To prevent these negative effects, protease and phosphatase inhibitors can be added to the lysis reagents. Numerous compounds have been identified that can inactivate or block the activities of proteases and phosphatases.

Protease and phosphatase inhibitor product features:

  • Convenient—ready-to-use, fully disclosed, broad-spectrum formulations available as either liquid cocktails, tablets, or capsules, in multiple pack sizes and with a minimum of 1 year of shelf life
  • Complete protection—all-in-one formulations containing both protease and phosphatase inhibitors are offered in both liquid and tablet formulations (with EDTA or EDTA-free)
  • Compatible—use directly with Thermo Scientific Pierce Cell Lysis Buffers, other commercial, or homemade detergent-based lysis reagents

Broad effective inhibition of proteases and phosphatases

Bar chart of percent inhibition for Pierce protease inhibitor mini tablets and tablets from 2 competitors

Performance comparison between three commercially available protease inhibitor tablets. Pancreatic extract (100 μL; 0.5 μg/μL) was incubated with quenched fluorescent protease-cleavable substrates for trypsin, cysteine, and metalloprotease and cathepsins, in the presence of the reformulated Thermo Scientific Pierce Protease Inhibitor Mini Tablets, Roche™ Complete™ Protease Inhibitor Tablets, and Sigma-Aldrich™ SIGMAFAST™ Protease Inhibitor Cocktail Tablets, with and without EDTA. Reactions were incubated for 1 hr at 37ºC, and fluorescence was determined at the appropriate emission wavelengths. The percent inhibition is shown for each protease inhibitor formulation.

Protein phosphorylation in cell extracts is broadly preserved by Thermo Scientific Phosphatase Inhibitor Mini Tablets. (A) HCT116 cells were serum-starved, then either treated with EGF for 15 min or left as control cells. Cell lysates were prepared in Thermo Scientific Pierce IP Lysis Buffer with Thermo Scientific Protease and Phosphatase Inhibitor Mini Tablets, EDTA-Free, or with no inhibitor. Lysate containing 500 μg of protein was then incubated with 5 μg of phospho-tyrosine antibody overnight at 4ºC. The complex was then incubated with Thermo Scientific Pierce Protein A/G Magnetic Beads for 1 hr at room temperature. Beads were washed, and low-pH elution was performed. The eluates were subjected to western blotting, and the membrane was then probed with EGFR antibody for chemiluminescence detection. (B) The degree of inhibition for protein, alkaline, and acid phosphatase activity was determined in kidney extract (25 μL; 0.5 μg/μL) by incubating extracts with a fluorogenic substrate (MFP or FDP) that measures phosphatase activity upon desphosphorylation in the presence of Pierce Phosphatase Inhibitor Mini Tablets, Roche™ PhosStop™ Phosphatase Inhibitor Tablets, and Sigma-Aldrich™ Phosphatase Inhibitor Cocktail 2 and 3 liquid formulations. Reactions were incubated for 1 hr at 37ºC, and fluorescence was determined at the appropriate emission wavelength. The percent inhibition is shown for each phosphatase inhibitor formulation.

Tips

  • Cell lysis disrupts cells membranes and organelles resulting in unregulated proteolytic activity that can reduce protein yield and function. To prevent extracted protein degradation, it is often necessary to add protease and phosphatase inhibitors to cell lysis reagents.
  • Most researchers use a mixture of several different inhibitor compounds to ensure the protein extracts do not degrade before analysis of the target of interest. Protease inhibitors are nearly always needed, while phosphatase inhibitors are required only when investigating phosphorylation states.
  • Analyze a sample of the solubilized protein and the insoluble fractions by SDS-PAGE to determine the efficiency of the protein extraction method used.
     

Protein extraction and stabilization products

Tissue and cell lysis

Product selection guides Products
Mammalian cell protein extraction T-PER Tissue Protein Extraction Reagent
N-PER Neuronal Protein Extraction Reagent
M-PER Mammalian Protein Extraction Reagent
RIPA Lysis Buffer
IP Lysis Buffer
Insect protein extraction I-PER Insect Cell Protein Extraction Reagent
Bacterial cell lysis B-PER Complete Bacterial Protein Extraction Reagent
B-PER Bacterial Protein Extraction Reagent
B-PER (PBS) Bacterial Protein Extraction Reagent
B-PER II (2X) Bacterial Protein Extraction Reagent
Plant protein extraction Plant Total Protein Extraction Kit
Yeast protein extraction Y-PER Yeast Protein Extraction Reagent
Subcellular fractionation NE-PER Nuclear and Cytoplasmic Extraction Reagents
Subcellular protein fractionation kits (tissue or cultured cells)
Syn-PER Synaptic Protein Isolation Kit
Membrane protein extraction and isolation GPCR Extraction and Stabilization Reagent
Mem-PER Plus Membrane Protein Extraction Kit
Cell Surface Protein Isolation Kit
Organelle isolation Lysosome Enrichment Kit for Tissues and Cultured Cells
Organelle isolation using magnetic beads
Mitochondrial isolation kits (tissue or cultured cells)
Neuronal cell protein extraction N-PER Neuronal Protein Extraction Reagent
Syn-PER Synaptic Protein Isolation Kit

Detergents for protein solubilization

Product selection guides Products
Detergents for protein solubilization Surfact-Amps detergents
n-Dodecyl-beta-maltoside detergent
CHAPS detergent (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate)
Sodium dodecyl sulfate (SDS)
Octylthioglucoside (OTG) detergent
Octyl-beta-glucoside detergent

Protease and phosphatase inhibitors

Product selection guides Products
Protease and phosphatase inhibitors Protease liquid cocktails, tablets, and capsules Halt Protease Inhibitor Cocktail
Halt Protease Inhibitor Cocktail, EDTA free
Pierce Protease Inhibitor tablet
Pierce Protease Inhibitor mini tablet
Pierce Protease Inhibitor tablets, EDTA-free
Pierce Protease Inhibitor mini tablets, EDTA-free
Pierce Protease Inhibitor XL Capsules, EDTA-free
Phosphatase liquid cocktail and tablets Halt Phosphatase Inhibitor Cocktail
Pierce Phosphatase Inhibitor Tablet
Combined protease and phosphatase liquid cocktails and tablets Halt Protease and Phosphatase Inhibitor Cocktail
Halt Protease and Phosphatase Inhibitor Cocktail, EDTA free
Pierce Protease and Phosphatase Inhibitor Mini Tablet
Pierce Protease and Phosphatase Inhibitor Mini Tablet, EDTA Free

Purify and enrich your protein sample

Various methods are used to enrich or purify a protein of interest from other proteins and components in a crude cell lysate or other sample. Ion exchange and affinity chromatography are two commonly used strategies for partial or 1-step purification.

Affinity purification

Also known as affinity chromatography, this purification method is enabled by the specific binding properties of a protein to an immobilized ligand. Since the protein of interest is tightly bound, contaminants can be removed through wash steps, and the bound protein can be stripped (eluted) from the support in a highly purified form. Affinity purification is desirable because it often produces higher protein yields and requires less steps than other purification methods. It is the method of choice for purifying recombinant or biotinylated proteins and antibodies.

Discover more about protein purification
See protein isolation and purification learning resource

Ion exchange (IEX) purification

Also known as ion exchange chromatography, this purification method enables the separation of proteins based on the protein charge at a particular pH. Since multiple proteins may have similar charges, IEX chromatography generally enables only partial purification of a protein of interest when used early in a multistep purification process. However, IEX resins can also be used during a final polishing step to remove specific contaminants that persist after other purification steps. Typically, proteins bind to the IEX column at low ionic strength and elute differentially by increasing salt concentration or changing pH in a gradient. A cation exchange resin binds to positively charged proteins; an anion exchange resin binds to negatively charged proteins. Ion exchange resins are classified as “weak” or “strong”, which refers to the extent that the ionization state of the functional groups varies with pH.

Discover more about ion exchange purification

How protein purification works

  1. Bind: Capturing protein of interest using a specific ligand.
  2. Wash: Remove unwanted proteins and other impurities, such as nucleic acids, during the wash phase.
  3. Elute: This step reverses the binding reaction. One can elute and enrich the target protein by varying buffering conditions such as pH, salt concentration, and detergents, or including competitive binders.
  4. Polishing: Using an additional chromatography step (usually ion-exchange or size exclusion) to remove remaining trace impurities and other contaminants by making best use of the known characteristics of the target protein.

Protein purification workflow

Chart of protein purity as a function of the number of purification steps in the purification method

Comparison of DYKDDDDK-tagged SUMO protein yield and background using Pierce anti-DYKDDDDK resin and other products C- and N-terminal DYKDDDDK-tagged SUMO proteins were expressed in E. coli and purified using Pierce Anti-DYKDDDDK Magnetic Agarose, Sigma-Aldrich® Anti-FLAG™ M2 Magnetic Beads, and MBL Anti-DDDDK-tag mAb-Magnetic Agarose. Tagged protein was competitively eluted with Pierce 3x DYKDDDDK Peptide, and the results were analyzed by SDS-PAGE (A) and densitometry using the Invitrogen iBright Imaging System (B). Comparison between the starting lysate and elution fractions shows effective immunoprecipitation and elution of DYKDDDDK-tagged protein, with minimal background from the Pierce magnetic agarose compared to the other suppliers’ products.

Protein purification product features:

  • Broad product selection—strong ion exchange and affinity supports for the purification and enrichment of proteins and antibodies; affinity ligands enable 1-step purification of recombinant and biotinylated proteins, while activated supports provide a platform for custom protein immobilization
  • High performance—resins are designed to maximize protein yield and reduce background
  • More formats—magnetic beads, loose resins, FPLC cartridges, and 96-well filter plates enable protein purification from screening and small-scale phases to process-scale purification
  • Economical—pricing that is similar to or better than other leading suppliers
Types of affinity purification Description
Protein immobilization
(Activated supports for custom immobilization)
Uses activated supports and accessories for the immobilization of proteins, antibodies, and other molecules. These resins or magnetic beads are available separately or in convenient kits. Different reactive chemistries are available to optimize immobilization based on the ligand properties.

See covalent immobilization of affinity ligands learning resource
Antibody purification Proteins A, G, A/G, and L have unique properties, which make each one suitable for different types of antibody targets (e.g., antibody subclass or animal species). These ligands enable purification of general immunoglobulins from a crude sample. Depending on the sample source, an antigen-specific antibody may account for only a small portion of the total immunoglobulin in the sample. For example, generally only 2–5% of total IgG in mouse serum is specific for the antigen used to immunize the animal.

See antibody purification learning resource
Recombinant protein purification
(Fusion protein purification)
Uses resins for the purification of recombinant proteins from cultures such as E. coli or Pichia. These resins are available in multiple formats to accommodate a variety of needs, from high-throughput screening to batch and pilot-scale purification. Superflow resins have undergone extensive chemical characterization. We have ligands targeting a variety of fusion tags, including 6xHis, GST, anti-DYKDDDDK (anti-FLAG™), c-Myc, and HA.

See fusion protein learning resource
Purification using magnetic Agarose Magnetic agarose beads consist of highly crosslinked agarose encapsulating a ferrimagnetic core. The beads are 10–40 uM in size and have higher binding capacity than traditional magnetic beads. We offer a variety of ligands for immunoprecipitation (IP), co-immunoprecipitation (co-IP), pull-down, and other high throughput affinity screening applications, utilizing immobilized Protein A/G, Ni-NTA, Glutathione, and Anti-DYKDDDDK. The beads are removed from the solution manually using a magnetic stand or by automation using an instrument such as the Thermo Scientific KingFisher Flex Magnetic Particle Processor. Automated instruments are especially useful for higher throughput purification and screening of purification conditions.

See scientific poster: High capacity magnetic supports for automated antibody and epitope-tagged protein purifications
Biotin affinity purification Uses resins for the purification of biotinylated or desthiobiotinylated proteins, peptides, and other molecules. These resins are available in multiple pack sizes, as well as in spin columns, kits, FPLC cartridges, and coated plates. Different biotin-binding ligands are available based on elution conditions or level of purity.

See avidin-biotin interaction learning resource
Immunoprecipitation (IP) Small-scale affinity purification of antigens using a specific antibody that is immobilized to a solid support such as magnetic beads or agarose resin. IP is one of the most widely used methods for isolation of proteins and other biomolecules from cell or tissue lysates for the purpose of subsequent detection by western blotting and other assay techniques.

See immunoprecipitation learning resource
See IP support
Co-immunoprecipitation (co-IP) and pull-down Similar to IP, except the target antigen precipitated by the antibody is used to co-precipitate its binding partner(s) or associated protein complex from the lysate and pull-downs. This method is used when antibodies to specific proteins are not available. These “bait” proteins are tagged with an epitope to which a high-affinity antibody is available and ectopically expressed in the cell of interest.

See co-immunoprecipitation learning resource
See pull-down assays learning resource
See co-IP and pull-down support
ChIP, RIP, and protein-nucleic acid pull-down Chromatin immunoprecipitation (ChIP) assays are performed to identify regions of the genome with which DNA-binding proteins, such as transcription factors and histones, associate. In ChIP assays, proteins bound to DNA are temporarily crosslinked and the DNA is sheared prior to cell lysis. The target proteins are immunoprecipitated along with the crosslinked nucleotide sequences, and the DNA is then removed and identified by PCR, sequenced, applied to microarrays, or analyzed in some other way.

RNA immunoprecipitation (RIP) uses an approach similar to ChIP, except that RNA-binding proteins are immunoprecipitated instead of DNA-binding proteins. Immunoprecipitated RNAs can then be identified by RT-PCR and cDNA sequencing.

See ChIP learning resource
See protein-nucleic acid interaction support
Endotoxin detection and removal kits Endotoxin is a type of pyrogen and is a component of the exterior cell wall of Gram-negative bacteria. It is an unwanted and toxic by-product of recombinant proteins purified from E coli. Therefore, its removal from protein samples is an important step for downstream applications.

See app note: Eliminate endotoxins from protein and antibody samples

Overview of ion exchange, affinity, and activated supports

Application Purity level Ligand and/or chemistry Base bead type Packaging options
Ion exchange purification Medium to high (aication-specific) Strong anion exchange POROS Loose resin
Strong cation exchange
Antibody purification High Protein A, protein G, protein A/G Agarose, magnetic beads, magnetic agarose, POROS Loose resins or beads, spin columns and kits, chromatography cartridges, 96-well spin plates
Protein L Agarose, magnetic beads
Melon Gel Agarose
Fusion protein purification High Ni-NTA, Ni-IDA, cobalt, glutathione Agarose, Superflow, magnetic beads, magnetic agarose Loose resins or beads, spin columns and kits, chromatography cartridges, 96-well spin plates
Anti-c-Myc, anti-HA, anti-FLAG Agarose, UltraLink magnetic beads Loose resins or beads, kits
Biotin affinity purification High Avidin, streptavidin, NeutrAvidin, monomeric avidin Agarose, magnetic beads Loose resins, spin columns and kits, chromatography cartridges, 96-well spin plates
Protein immobilization High Amine-reactive, sulfhydryl-reactive, carbonyl-reactive, carboxyl-reactive Agarose Loose resins or dry powder
Epoxy, tosyl-activated, carboxylic acid, amine Magnetic beads Loose beads

Select your resin based on purification scale and application

Scale High-throughput screening High-throughput batch Batch Pilot Process
Description Small scale, automation compatible Lab or bench scale Lab or bench scale Scale-up desired Production scale
Yield Microgram Milligram Milligram Gram Kilogram
Format Magnetic particle processor Magnetic particle processor, 96-well spin plate (agarose) Gravity flow, spin column (agarose), fast protein liquid chromatography (FPLC) at low flow rates FPLC at medium flow rates FPLC at high flow rates
Application High-throughput screening, interaction studies (IP, co-IP, pull-down), mutational analysis High-throughput screening, interaction studies (IP, co-IP, pull-down), mutational analysis requiring mg scale Functional assays, structural analysis Structural analysis, intermediate-scale production Bulk production
Recommended resin type Magnetic bead (1-2.8 µm)  
  Magnetic agarose (10-40 µm)  
  Agarose (45-165 µm)  
  Superflow (45-165 µm)  
  UltraLink resin (50-80 µm)  
  POROS resin (50 µm)

Tips

The process of protein purification varies depending on the downstream analyses to be performed. Certain steps may be repeated or omitted to achieve the desired result.

Purification products

Protein immobilization

Product selection guides Products
Protein immobilization Pierce NHS-Activated Agarose
AminoLink Coupling Resin
AminoLink Plus Coupling Resin
SulfoLink Coupling Resin
GlycoLink Coupling Resin
CarboxyLink Coupling
Additional activated supports and accessories Pierce NHS-Activated Agarose Spin Columns
AminoLink Plus Immobilization Kit
AminoLink Plus Micro Immobilization Kit
AminoLink Immobilization Kit
AminoLink Reductant
Pierce CDI-activated Agarose Resin
SulfoLink Immobilization Kit for Peptides
UltraLink Iodoacetyl Micro Peptide Coupling Kit
GlycoLink Immobilization Kit
GlycoLink Micro Immobilization Kit
CarboxyLink Immobilization Kit

Antibody purification

Product selection guides Products
Antibody purification Protein A Magnetic beads Pierce Protein A Magnetic Beads
Dynabeads Protein A Magnetic Beads
Resins Pierce Protein A Agarose
Pierce Protein A Plus Agarose
Pierce Recombinant Protein A Agarose
POROS MabCapture A Select
Spin columns and kits NAb Protein A Plus Spin Columns
NAb Protein A Plus Spin Kits
Buffers Pierce Protein A IgG Binding Buffer
Protein G Magnetic beads Pierce Protein G Magnetic Beads
Dynabeads Protein G Magnetic Beads
Resins Pierce Protein G Agarose
Pierce Protein G Plus Agarose
POROS MabCapture G Select
Spin columns and kits NAb Protein G Spin Columns
NAb Protein G Spin Kits
Pierce Recombinant Protein G
Buffer Pierce Protein G IgG Binding Buffer
Protein A/G Magnetic beads Pierce Protein A/G Magnetic Agarose Beads
Pierce Protein A/G Magnetic Beads
Resins Pierce Protein A/G Agarose
Pierce Protein A/G Plus Agarose
POROS MabCapture A/G Select
Spin columns and kits NAb Protein A/G Spin Columns
NAb Protein A/G Spin Kit
Pierce Recombinant Protein A/G
Buffer Pierce Protein A/G IgG Binding Buffer
Protein L Magnetic beads Pierce Protein L Magnetic Beads
Resins Pierce Protein L Agarose
Pierce Protein L Plus Agarose
Spin columns and kits NAb Protein L Spin Columns
NAb Protein L Spin Kit
Pierce Recombinant Protein L
Melon Gel Melon Gel IgG Spin Purification Kit
Melon Gel IgG Purification Kit
Melon Gel Monoclonal IgG Purification Kit
Monoclonal IgG Purification Kit
Melon Gel Spin Plate Kit for IgG Screening
Melon Gel Chromatography Cartridges (1 mL or 5 mL)
Melon Gel Regenerant
Ascites Conditioning Reagent for Melon Gel
Melon Gel Purification Buffer
Buffers and related products Pierce IgG Elution Buffer
Pierce Gentle Ag/Ab Binding Buffer, pH 8.0
Pierce Gentle Ag/Ab Elution Buffer, pH 6.6
Pierce Gentle Ag/Ab Binding and Elution Buffer Kit
Pierce Saturated Ammonium Sulfate Solution
Pierce Thiophilic Adsorption Kit
Pierce Mannan Binding Protein Agarose
Pierce IgM Purification Kit
Elution Buffer for Pierce IgM Purification Kit
MBP Column Preparation Buffer for Pierce IgM Purification Kit
Pierce Jacalin Agarose

Recombinant protein purification

Product selection guides Products
His-tagged (6xHis) protein purification Pierce Ni-NTA Magnetic Beads
Pierce Ni-NTA Magnetic Agarose Beads
HisPur Ni-NTA Agarose Resin        
HisPur Ni-NTA Superflow Resin
HisPur Cobalt Agarose Resin
HisPur Cobalt Superflow Resin
GST-tagged protein purification Pierce Glutathione Magnetic Agarose Beads
Pierce Glutathione Agarose Resin
Pierce Glutathione Superflow Resin
Other epitope-tagged protein purification Pierce Anti-DYKD4K (FLAG) Magnetic Agarose
Pierce Anti-DYKD4K (FLAG) Affinity Resin UltraLink
Pierce Anti-c-Myc Magnetic Beads
Pierce Anti-c-Myc Agarose (Superflow 6)
Pierce Anti-HA Magnetic Beads
Pierce Anti-HA Agarose
Purification using magnetic agarose Pierce Ni-NTA Magnetic Agarose Beads
Pierce Glutathione Magnetic Agarose Beads
Pierce Anti-DYKD4K (FLAG) Magnetic Agarose Beads

Biotin affinity purification

Product selection guides Products
Biotin affinity purification Magnetic beads Pierce Streptavidin Magnetic Beads
Resins Pierce Avidin Agarose
Pierce Streptavidin Agarose
Pierce Streptavidin Agarose HC
Pierce NeutrAvidin Agarose
Pierce NeutrAvidin Agarose HC
Pierce Monomeric Avidin Agarose
Related products for biotin-binding applications Invitrogen CaptAvidin Agarose (Sedimented Bead Suspension)
Pierce Streptavidin Agarose Columns
Pierce High Capacity Streptavidin Chromatography Cartridge
Pierce Monomeric Avidin Agarose Kit
Pierce Biotin
Pierce Iminobiotin Agarose

IP, co-IP, pull-down

Product selection guides Products
Immunoprecipitation (IP) Antibody-binding IP products Protein A, G, A/G Magnetic beads
  • Dynabeads Protein A
  • Dynabeads Protein G
  • Pierce Protein A/G
Kits
  • Dynabeads Protein A kit
  • Dynabeads Protein G kit
  • Pierce Protein A/G kit
  • Pierce Crosslink Kit (A/G)
Secondary antibodies (anti-mouse, anti-rabbit) Magnetic beads
  • Dynabeads M-280, Sheep-anti Mouse IgG
  • Dynabeads M-280, Sheep-anti Rabbit IgG
Surface-activated beads (epoxy) Kits
Biotin-binding IP products
  • Pierce Streptavidin Magnetic Beads
  • Dynabeads M-280 Streptavidin
  • Dynabeads M-270 Streptavidin
  • Dynabeads MyOne Streptavidin C1
  • Dynabeads MyOne Streptavidin T1
  • DynaMag-2 Magnet
Recombinant protein (fusion tag) IP products
  • HisPur Ni-NTA Magnetic Beads
  • Pierce Ni-NTA Magnetic
  • Agarose Beads
  • Dynabeads His-Tag Isolation and Pulldown
  • Pierce Glutathione Magnetic Agarose Beads
  • Pierce Anti-DYKDDDDK Magnetic Agarose
  • Pierce Anti-HA Magnetic Beads
  • Pierce HA-Tag Magnetic IP/Co-IP Kit
  • Pierce Anti-c-Myc Magnetic Beads
  • Pierce c-Myc-Tag Magnetic IP/Co-IP Kit
  • DynaMag-2 Magnet
Co-immunoprecipitation (co-IP) and pull-down Spin column
Magnetic beads
ChIP, RIP, and Protein-Nucleic Acid Pull-Down ChIP
  • MAGnify Chromatin Immunoprecipitation System
  • Pierce Magnetic ChIP Kit
  • Pierce ChIP-grade Protein A/G Magnetic Beads
  • Proteinase K Solution, ChIP grade
RIP
  • Pierce Magnetic RNA-Protein Pull-Down Kit
  • Pierce Streptavidin Magnetic Beads
  • Pierce RNA 3' End Desthiobiotinylation Kit

Ion exchange purification

Product selection guides Products
Ion exchange purification Ion exchange spin columns Pierce (SCX) Spin Columns
Pierce (SAX) Spin Columns
Ion Exchange Purification
Ion exchange resins POROS XS Resin
POROS XQ Resin
POROS 50 HQ Resin

Endotoxin removal

Product selection guides Products
Endotoxin detection Pierce Chromogenic Endotoxin Quant Kit
Pierce LAL Chromogenic Endotoxin Quantitation Kit
Endotoxin removal Loose resin
Spin columns (0.25 mL, 0.5 mL, or 1 mL)

Clean up your protein sample

Many detergents and salts used in protein extraction formulations may have adverse effects on protein function or stability, or may interfere with downstream analysis. Therefore, it may be necessary to remove or reduce these contaminants following cell lysis or subsequent sample processing such as protein purification.

Download Protein Clean-Up Technical Handbook

Discover more about protein dialysis, desalting, and concentration

Tips

  • If the protein concentration is too dilute for further processing or analysis, the sample can be concentrated quickly using centrifugal concentrators.
  • For buffer exchange during concentration technique, the retentate can be diluted with exchange buffer and centrifuged. This process can be repeated until the desired level of exchange or desalting has been achieved.

Protein clean-up products

Product category Products
Protein dialysis Slide-A-Lyzer products
Protein desalting Zeba products
Protein concentration Protein concentrators
Detergent removal products HiPPR Detergent Removal 96-well Spin Plates
Abundant protein depletion High Select Top 14 and Top 2 reagents
Small molecule removal Biotin, dye, crosslinkers, and reducing agent removal products

Protein dialysis

Dialysis is a classic clean-up technique that removes small molecules and unwanted compounds by selective diffusion through a semi-permeable membrane. A sample and a buffer solution are placed on opposite sides of the membrane. Proteins that are larger than the membrane pores are retained on the sample side of the membrane, but smaller molecules (contaminants) diffuse freely through the membrane until an equilibrium concentration is achieved. Through this technique, the concentration of small contaminants in the sample can be decreased to acceptable levels.

Protein dialysis products features:

  • Excellent sample recovery—low-binding plastics and membranes help minimize sample loss compared to filtration and resin systems
  • Convenience—easy-to-grip design helps simplify sample addition and removal with syringe and/or pipette
  • Secure—sealed membranes help prevent leakage that can occur with dialysis tubing and homemade devices
  • Validated—each device is be leak-tested during production

Discover more about protein dialysis
See protein dialysis learning resource

Product highlight

MWCO* membrane 10-100 µL
Pierce 96-well Microanalysis Plate
10-2,000 µL
Slide-A-Lyzer MINI Dialysis Device
0.1-70 mL
Slide-A-Lyzer G2 Dialysis Cassette
0.1-30mL
Slide-A-Lyzer Dialysis Cassette
150-250 mL
Slide-A-Lyzer Dialysis Flask
15-100 mL
SnakeSkin Dialysis Tubing
2K N/A
See product
N/A
3.5K
See product

See product
7K N/A N/A
10K
See product

See product
20K N/A
See product
N/A
See full comparison of these products ›

*MWCO: Molecular weight cut off.

Protein recovery by molecular weight cutoff (MWCO)

Sample retention by the 2K, 3.5K, 7K, 10K, and 20K MWCO Thermo Scientific Slide-A-Lyzer cassette membrane. Individual proteins or vitamin B12 (1 mg/mL) in either saline or 0.2 M carbonate-bicarbonate buffer, pH 9.4 were dialyzed overnight (17 hours) at 4°C. The amount of retentate was estimated using either the Pierce BCA Protein Assay Kit or absorption at 360 nm (for vitamin B12).

Dialysis rates for various formats

The rate of removal of NaCl using various dialysis products. NaCl removal from samples was determined by measuring the conductivity of the retentate at the indicated times. (A) Slide-A-Lyzer MINI Dialysis Device (10K MWCO, 2 mL) versus conventional dialysis. Bovine serum albumin (BSA) samples (2 mL, 0.25 mg/mL in 1 M NaCl) were dialyzed against 45 mL of water in 50 mL disposable conical tubes on an orbital shaker (300 rpm) at room temperature. The water was changed once after 2 hours. Results are the average of two samples. For conventional dialysis, the samples were dialyzed against 2 L of water in a beaker with stirring. Greater than 95% of NaCl was removed within 4 hours. (B) Samples of 0.1 mL (0.4 mg/mL cytochrome C containing 1 M NaCl) were dialyzed in the Pierce 96-well Microdialysis Plate against 1.8 mL of water at RT with gentle shaking. The buffer was changed at 1-, 2-, and 3-hour intervals over a 4-hour period. Removal of NaCl was >83% after 2 hours and >99% after 4 hours. (C) Proteins in 200 mL samples containing 1 M NaCl were dialyzed at room temperature using Slide-A-Lyzer Dialysis Flasks with 2K, 3.5K, 10K, and 20K MWCOs. The dialysis buffer (4 L) was changed after 2 and 5 hours (triangles; also at 41 hours for the 2K condition). Greater than 95% of NaCl was removed within 8 to 18 hours (41 hours for the 2K condition).

Protein desalting

Size exclusion chromatography, also described as gel filtration, can be used for removal of salts from samples. Using this technique, a resin is selected with pores large enough for salts to penetrate, but small for the protein of interest to enter. This causes contaminants to slow down their rate of migration. The larger and faster proteins separate from the slower and smaller molecules during gravity flow or centrifugation.

Discover more about protein desalting
See protein desalting learning resource

Protein desalting products features:

  • High performance—proprietary resin enables excellent protein recovery and efficient contaminant removal
  • Flexible—available in spin columns, filter spin plates, and cartridges for a range of needs
  • Fast—no fraction screening or waiting for protein to emerge by gravity flow
  • Economical—cost-effective products that offer great performance

Product highlight

Type Spin columns Spin plates Chromatography columns
Format Micro 0.5 mL 2 mL 5 mL 10 mL 96-well 1 mL 5 mL
Resin bed 75 µL 0.5 mL 2 mL 5 mL 10 mL 550 µL 1 mL 5 mL
Sample volume (7K MWCO) 2-12 µL 30-130 µL 200-700 µL 500-2,000 µL 700-4,000 µL 20-100 µL 50-250 µL 100-1,500 µL
Sample volume (40K MWCO) 5-14 µL 70-200 µL 200-900 µL 300-2,000 µL 1,000-4,000 µL 20-100 µL N/A N/A
See full comparison of these products ›

Comparison of protein recovery and sample dilution

Zeba Spin Desalting Columns result in a high protein recovery while providing minimal sample dilution over a wider range of sample concentrations and volumes compared to alternative products. Zeba Spin Desalting Columns, 10 mL (7K MWCO) and GE PD-10 Columns were used to desalt 1.5, 2.5, and 3.5 mL BSA samples at a concentration of 0.04, 0.2, and 1 mg/mL. Desalting was performed according to the manufacturers’ recommended protocols; both the spin and gravity protocols were used for the GE PD-10. Protein recovery was analyzed by SDS-PAGE. For each electrophoresis gel, an aliquot of starting sample equal to 1 μg of BSA was loaded in lane 1 as the loading control; all other desalted samples were loaded in the gel at the same volume as the loading control. Differences in intensity between lanes are a combination of protein recovery and sample dilution caused by desalting. The largest differences in recovery and concentration are noted in the highlighted area.

Protein concentrators

Protein concentration is similar to dialysis and uses a semi-permeable membrane to separate proteins from low molecular weight compounds. Unlike dialysis, which relies on passive diffusion, concentration is achieved by forcing solution through membrane using centrifugation. During centrifugation, both buffer and low molecular weight solutes are forced through the membrane where they are collected on the other side (filtrate). Macromolecules (proteins) remain on the sample side of the membrane, where they become concentrated to a smaller volume (retentate), as the reagent is forced across the membrane to the other side.

Discover more about protein concentrators
See protein dialysis, desalting, and buffer exchange learning resource

Protein concentrator product features

  • Rapid processing—unique design minimizes membrane fouling; and sample concentration of 10- to 30-fold can be achieved in 5–30 minutes for 10K MWCO (device-dependent times may vary for other MWCOs), even with particle-laden solutions
  • High recovery—retain >90% of protein samples while removing contaminants or exchanging buffers
  • Convenient—clear markings, wide sample chamber, and removable filtrate chamber make handling simple and easy
  • Instrument compatible—can be used with standard centrifuges utilizing either fixed-angle or swinging-bucket rotors

Product highlight

Volume range 0.1–0.5 mL 2–6 mL 5–20 mL 20–100 mL
MWCOs available 3K, 10K, 30K, 100K 3K, 10K, 30K, 100K 3K, 10K, 30K, 100K 5K, 10K, 30K, 100K
Processing time* 3-15 min 15-90 min 15-60 min 15-90 min
Retentate volume range* 9-67 µL 51-174 µL 121-777 µL 1.9-3.5 mL
Protein recovery range* 95-100% 94-100% 94-100% 92-98%
See full comparison of these products ›

*Four different protein solutions were used for each MWCO

Comparison of protein recovery between Pierce Protein Concentrators (using 3K, 5K, 10K, 30K, or 100K MWCO) and other vendors for 0.5 mL, 6 mL, 20 mL, and 100 mL concentrators. Samples of different protein solutions were centrifuged in Pierce Protein Concentrators and other suppliers’ concentrators according to manufacturers’ instructions: 0.5 mL (15,000 x g), 5 mL (4,000 x g), 20 mL (4,700 x g), and 100 mL (1,200 x g). Samples were centrifuged until a greater than 15- to 30-fold decrease in sample volume was achieved; protein concentration was measured by either Pierce BCA Protein Assay Kit (0.5 mL concentrators only) or absorbance at A280.

Quantify and detect your target protein

There are various methods that can be used to detect and measure your target protein depending on your experimental needs. Below are common techniques used to detect and measure proteins from complex mixtures (e.g., lysates, sera) and the typical requirements for each method.

Total protein quantitation

Quantifying total protein concentration is an important step in workflows involving isolation, separation, and analysis of proteins by biochemical methods. Assay methods may use colorimetric or fluorescent detection with fluorometers, spectrophotometers, or plate readers. Every protein assay has limitations depending on the application and the specific protein sample analyzed. The most useful features to consider when choosing a protein assay are sensitivity (lower detection limit), compatibility with common substances in samples (e.g., detergents, reducing agents, chaotropic agents, inhibitors, salts, and buffers), standard curve linearity, and protein-to-protein variation.

Colorimetric protein assays

Colorimetric signals can be detected using a microplate reader or spectrophotometer. The most popular colorimetric protein assays are:

  • BCA Assays—Protein-copper chelation with secondary detection of the reduced copper.
  • Bradford Assays—Protein-dye binding with direct detection of the color change associated with the bound dye.
     

Fluorescent protein assays

Fluorescence-based protein quantitation is an alternative to colorimetric methods. Fluorescence detection methods offer excellent sensitivity, requiring less protein sample thereby leaving more sample available for your experiment. Additionally, read time is not a critical factor, so the assays can be readily adapted for automated high-throughput applications. The fluorescence signal can be detected using a fluorometer or microplate reader.

Discover more about protein assays and analysis
See protein quantitation and detection learning resource
Learn more about methods and technologies for identifying and measuring proteins

Product highlight Category Description
Pierce BCA Protein Assay Kit Colorimetric Two-component, high-precision, detergent-compatible protein assay. Compared to most dye-binding methods, the BCA assay is affected much less by protein compositional differences, providing greater concentration accuracy.
Pierce Rapid Gold BCA Protein Assay Kit Colorimetric This kit maintains the key characteristics of the traditional BCA assay but allows a fast time and room temperature incubation equal to dye-binding methods.
Quant-iT Protein Assay Fluorescence The assay is highly selective for protein and exhibits very little protein-to-protein variation. The assay is performed at room temperature, and the signal is stable for 3 hours.
Qubit 4 Fluorometer Fluorometer The Qubit 4 Fluorometer is the latest version of the popular Qubit fluorometer designed to accurately measure protein quantity.

Use the interactive Protein Assay Selection Guide

Tips

  • No one reagent is the ideal or best protein assay method. Each method has its advantages and disadvantages.
    Use the interactive Protein Assay Selection Guide to filter products based on sample type, assay time, read-out (colorimetric or fluorescent), and compatibility to detergents or reducing agents
  • To learn about the different ways to quantitate protein concentrations continue reading the article: Overview of protein assays.

Protein detection

Protein detection method attributes ELISA Western blotting Mass spec
Advantages
  • High-throughput capability with 96-well or 384-well plate formats
  • Quantification of target proteins
  • Identification and verification of molecular weight
  • Ability to separate and isolate protein of interest
  • Identification and quantitation of multiple targets from the same sample
  • Detection of post-translational modifications or different isotypes
Sensitivity <5-10 pg/mL low femtogram to high attogram* attomolar range (1018)
Lysis buffer compatibility
  • For non-activity based ELISAs: ionic detergent based lysis buffers
  • For activity based ELISAs: non-ionic detergent-based lysis buffers (e.g. NP-40, Triton X-100)
  • For SDS-PAGE (denaturing): RIPA or other lysis buffers with ionic detergents
  • For native-PAGE applications: non-ionic detergents-based lysis buffers (e.g. NP-40, Triton X-100)
Detergents and high salts must be removed prior to analysis
Typical total protein required 0.1 µg/mL - 1 µ g/mL 1 µg- 50 µg <1 µg
Equipment required Plate reader X-ray film or CCD imaging equipment Mass spectrometer

*With high sensitivity HRP substrates, such as SuperSignal West Atto Ultimate Sensitivity Substrate

Protein quantitation and detection products

Choosing the right expression system for your production needs

Researchers have many methodology choices when it comes to producing recombinant proteins for early-stage discovery research through large-scale production of biotherapeutic drugs, vaccine development, and structural studies. It is imperative to use the right protein expression system for the target protein and application of interest. We offer a wide selection of superior mammalian, insect, bacterial, and yeast protein expression systems to suit your research needs.

Our Gibco Expi Transient Expression Systems, available in mammalian (CHO-S, 293F cells) and insect (Sf9 cells) expression formats, are completely optimized systems that enable rapid, high-yield production of proteins.

Icon-based chart showing the steps of protein expression from cells to purified protein
Bar charts of titer vs expression system for 3 different proteins

Recombinant protein titers in ExpiCHO and other transient CHO expression systems. Expression levels of human IgG, rabbit IgG and erythropoietin in ExpiCHO and other transient CHO expression systems are shown. ExpiCHO titers range from 25–160 times those of other transient CHO expression systems.

Product highlight Description
ExpiCHO Expression System The Gibco ExpiCHO Expression System is a completely optimized system consisting of ExpiCHO-S(TM) cells that have been adapted to high-density, serum-free suspension culture in ExpiCHO(TM) Expression Medium, along with specially designed transfection reagents and enhancers, that provide the highest yields possible in a transient system (up to 3g/L). That means you can start your research work in CHO cells and stay in CHO cells through discovery.
Expi293 Expression System The Gibco Expi293 Expression System is a completely optimized system consisting of Expi293F(TM) cells that have been adapted to high-density, serum-free suspension culture in Expi293(TM) Expression Medium, along with specially designed transfection reagents and enhancers, that enable recombinant 293-derived recombinant protein expression in just five to seven days, with a 2-10-fold increase in protein yields compared to previous generation transient expression systems
ExpiSf Expression System The ExpiSf Expression System is the first-ever chemically defined baculovirus-insect cell protein expression system, delivering superior yields (3x more protein) and consistent performance run after run using a fast, streamlined workflow.

Tips

Make sure to check for the following things when getting started with an Expi transient expression system:

  • Check your DNA: 260:280 ratio, 260:230 ratio, and the DNA concentration prior to transfection
  • Check your reagent storage conditions: 4-8°C and protected from light
  • Check your incubator: ensure display temperature, CO2%, and humidity are accurate and at 37°C, 8%, and 80% respectively
  • Check your cells: ensure doubling time, viability and cell diameter are all ideal and ready for transfection
  • Check your volumes: make sure you have the culture volume: flask size ratio of 1:3.6 (1:3 – 1:4 is ok)
  • Check your speed: decrease speed with increased volume; increase speed with decreased volume
Discover more about protein expression


Protein expression products

Product selection guides Products
Transient protein expression systems Expi293
ExpiCHO
ExpiSf Expression Systems
Mammalian protein expression Expi293
ExpiCHO Expression Systems
Insect protein expression ExpiSf Expression System
Bacterial protein expression Champion pET Expression System
Yeast protein expression PichiaPink Yeast Expression System
Structural Biology and Membrane Protein Expression Expi293 GnTI-
Expi293 Inducible
Expi293 Inducible GnTI- Expression Systems
Expi293 Met(-) Protein Labeling Kit
Protein expression selection guide Protein Expression Systems Selection Guide
Protein expression services Gene-to-Protein
Baculovirus Expression
Mammalian Expression services

Extract and stabilize your target protein from the sample

Protein extraction techniques vary depending on the source of the starting material, the location of the protein of interest within the cell, and the downstream application. Other important considerations include the preservation of protein activity and function as well as the reduction of background effects.

Protein extraction

Tissue and cell lysis

Historically, mechanical disruption has been used to lyse cells and tissues; our gentle, detergent-based solutions have been developed to efficiently lyse cells and enable the separation of subcellular structures without requiring physical disruption, providing high yields of active proteins.

Protein extraction product features:

  • Optimized—formulations maximize protein yield and preserve protein activity
  • Efficient—only produces minimal cross-contamination between subcellular fractions
  • Compatible—extracts can be used directly in most downstream applications
  • Gentle—eliminates the need for mechanical cell disruption for most sample types

High protein yield from a variety of mammalian cell types and cellular compartments

Western blot results to identify 7 different cellular proteins from 4 cell line lysates and 2 primary cell lysates

Protein extraction efficiency from major cellular compartments using M-PER Mammalian Protein Extraction Reagent. Lysates from established cell lines and primary cultures were prepared using M-PER reagent and extraction efficiency from the various cellular compartments evaluated. For each target protein, 10 µg of lysate was loaded for and electrophoresed by SDS PAGE, transferred to nitrocellose membrane and detected by western blot using SuperSignal West Pico PLUS Chemiluminescent Substrate.

Bar chart of protein yield per 10e6 cells vs cell line for 4 cell line lysates and 2 primary cell lysates

Protein yield from various cell types using M-PER Mammalian Protein Extraction Reagent. Cells were harvested at 85% confluency, washed twice and collected in ice-cold PBS and counted. For each cell type, 1 x 106 cells were pelleted by centrifugation at 2,000 x g for 5 minutes and lysed in 1 mL M-PER Reagent for 5 minutes. The cell lysates were clarified by centrifugation at 14,000 x g for 10 minutes and the supernatant was collected and the protein concentration (µg/million cells) was determined using the Pierce BCA Protein Assay.

Efficient and selective enrichment of membrane proteins

Improved protein yield using the Thermo Scientific Mem-PER Plus Membrane Protein Extraction Kit. Membrane proteins were isolated from mouse liver tissue and HeLa cells using Thermo Scientific Mem-PER Plus Membrane Protein Extraction Kit and three other commercial extraction kits. Protein yields (μg) for membrane, cytosolic, and total fractions were determined using the Thermo Scientific Pierce BCA Protein Assay Kit.

Efficient protein extraction from a variety of tissue types

Efficient extraction from bacterial cells

Protein yield comparison of two bacterial cell lysis reagents. E. coli ER2566/pLATE51-Klenow, ER2566/pGST-CC-StpB, and ER2566/pGS-Syk cell pellets (0.5 g), were resuspended in 2.5 mL aliquots of Thermo Scientific B-PER Complete Bacterial Protein Extraction Reagent or EMD Chemicals BugBuster Master Mix with gentle vortexing for 15 minutes at room temperature. Insoluble cell debris was removed by centrifugation at 16,000 x g for 20 minutes at 4°C. Protein yields (concentrations) for soluble fractions were determined using the Pierce BCA Protein Assay Kit.

Sample type Goal Product highlight
Primary or cultured mammalian cells or tissues Total protein extraction M-PER reagent
T-PER reagent
N-PER reagent
RIPA Lysis and Extraction Buffer
IP Lysis Buffer
Cultured mammalian cells or tissues Subcellular fractionation or organelle isolation NE-PER reagent
Subcellular Fractionation Kits
Mitochondria Isolation Kits
GPCR Extraction and Stabilization Reagent
Cell Surface Protein Isolation Kit
Syn-PER Reagent
Lysosome Enrichment Kit
Bacterial cells Total protein extraction B-PER reagent  
Yeast cells Total protein extraction Y-PER reagent  
Insect cells (baculovirus) Total protein extraction I-PER reagent  
Plant tissue (leaf, stem, root, flower) Total protein extraction Plan Total Protein Extraction Kit  

Detergent solutions

Detergents are frequently used in cell lysis reagent formulation and other protein research methods. Thermo Scientific Surfact-Amps Detergent Solutions are highly purified, precisely diluted (10%) formulations that are ideal for applications or assays that are sensitive to contaminants present in unpurified detergents.

Protein detergent product features:

  • Accurate—precise 10% detergent solution in ultrapure water
  • Easy-to-use—solution is simple to dispense and dilute
  • Exceptionally pure—less than 1.0 μq/mL peroxides and carbonyls
  • Stable—packaged under inert nitrogen gas in glass ampules or HDPE bottles
Graphic of simplified detergent molecule showing hydrophobic tail and hydrophilic head

Generic structure of a detergent molecule.

Protein stabilization

Cell lysis disrupts cell membranes and organelles, resulting in unregulated enzymatic activity that can reduce protein yield and function. To prevent these negative effects, protease and phosphatase inhibitors can be added to the lysis reagents. Numerous compounds have been identified that can inactivate or block the activities of proteases and phosphatases.

Protease and phosphatase inhibitor product features:

  • Convenient—ready-to-use, fully disclosed, broad-spectrum formulations available as either liquid cocktails, tablets, or capsules, in multiple pack sizes and with a minimum of 1 year of shelf life
  • Complete protection—all-in-one formulations containing both protease and phosphatase inhibitors are offered in both liquid and tablet formulations (with EDTA or EDTA-free)
  • Compatible—use directly with Thermo Scientific Pierce Cell Lysis Buffers, other commercial, or homemade detergent-based lysis reagents

Broad effective inhibition of proteases and phosphatases

Bar chart of percent inhibition for Pierce protease inhibitor mini tablets and tablets from 2 competitors

Performance comparison between three commercially available protease inhibitor tablets. Pancreatic extract (100 μL; 0.5 μg/μL) was incubated with quenched fluorescent protease-cleavable substrates for trypsin, cysteine, and metalloprotease and cathepsins, in the presence of the reformulated Thermo Scientific Pierce Protease Inhibitor Mini Tablets, Roche™ Complete™ Protease Inhibitor Tablets, and Sigma-Aldrich™ SIGMAFAST™ Protease Inhibitor Cocktail Tablets, with and without EDTA. Reactions were incubated for 1 hr at 37ºC, and fluorescence was determined at the appropriate emission wavelengths. The percent inhibition is shown for each protease inhibitor formulation.

Protein phosphorylation in cell extracts is broadly preserved by Thermo Scientific Phosphatase Inhibitor Mini Tablets. (A) HCT116 cells were serum-starved, then either treated with EGF for 15 min or left as control cells. Cell lysates were prepared in Thermo Scientific Pierce IP Lysis Buffer with Thermo Scientific Protease and Phosphatase Inhibitor Mini Tablets, EDTA-Free, or with no inhibitor. Lysate containing 500 μg of protein was then incubated with 5 μg of phospho-tyrosine antibody overnight at 4ºC. The complex was then incubated with Thermo Scientific Pierce Protein A/G Magnetic Beads for 1 hr at room temperature. Beads were washed, and low-pH elution was performed. The eluates were subjected to western blotting, and the membrane was then probed with EGFR antibody for chemiluminescence detection. (B) The degree of inhibition for protein, alkaline, and acid phosphatase activity was determined in kidney extract (25 μL; 0.5 μg/μL) by incubating extracts with a fluorogenic substrate (MFP or FDP) that measures phosphatase activity upon desphosphorylation in the presence of Pierce Phosphatase Inhibitor Mini Tablets, Roche™ PhosStop™ Phosphatase Inhibitor Tablets, and Sigma-Aldrich™ Phosphatase Inhibitor Cocktail 2 and 3 liquid formulations. Reactions were incubated for 1 hr at 37ºC, and fluorescence was determined at the appropriate emission wavelength. The percent inhibition is shown for each phosphatase inhibitor formulation.

Tips

  • Cell lysis disrupts cells membranes and organelles resulting in unregulated proteolytic activity that can reduce protein yield and function. To prevent extracted protein degradation, it is often necessary to add protease and phosphatase inhibitors to cell lysis reagents.
  • Most researchers use a mixture of several different inhibitor compounds to ensure the protein extracts do not degrade before analysis of the target of interest. Protease inhibitors are nearly always needed, while phosphatase inhibitors are required only when investigating phosphorylation states.
  • Analyze a sample of the solubilized protein and the insoluble fractions by SDS-PAGE to determine the efficiency of the protein extraction method used.
     

Protein extraction and stabilization products

Tissue and cell lysis

Product selection guides Products
Mammalian cell protein extraction T-PER Tissue Protein Extraction Reagent
N-PER Neuronal Protein Extraction Reagent
M-PER Mammalian Protein Extraction Reagent
RIPA Lysis Buffer
IP Lysis Buffer
Insect protein extraction I-PER Insect Cell Protein Extraction Reagent
Bacterial cell lysis B-PER Complete Bacterial Protein Extraction Reagent
B-PER Bacterial Protein Extraction Reagent
B-PER (PBS) Bacterial Protein Extraction Reagent
B-PER II (2X) Bacterial Protein Extraction Reagent
Plant protein extraction Plant Total Protein Extraction Kit
Yeast protein extraction Y-PER Yeast Protein Extraction Reagent
Subcellular fractionation NE-PER Nuclear and Cytoplasmic Extraction Reagents
Subcellular protein fractionation kits (tissue or cultured cells)
Syn-PER Synaptic Protein Isolation Kit
Membrane protein extraction and isolation GPCR Extraction and Stabilization Reagent
Mem-PER Plus Membrane Protein Extraction Kit
Cell Surface Protein Isolation Kit
Organelle isolation Lysosome Enrichment Kit for Tissues and Cultured Cells
Organelle isolation using magnetic beads
Mitochondrial isolation kits (tissue or cultured cells)
Neuronal cell protein extraction N-PER Neuronal Protein Extraction Reagent
Syn-PER Synaptic Protein Isolation Kit

Detergents for protein solubilization

Product selection guides Products
Detergents for protein solubilization Surfact-Amps detergents
n-Dodecyl-beta-maltoside detergent
CHAPS detergent (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate)
Sodium dodecyl sulfate (SDS)
Octylthioglucoside (OTG) detergent
Octyl-beta-glucoside detergent

Protease and phosphatase inhibitors

Product selection guides Products
Protease and phosphatase inhibitors Protease liquid cocktails, tablets, and capsules Halt Protease Inhibitor Cocktail
Halt Protease Inhibitor Cocktail, EDTA free
Pierce Protease Inhibitor tablet
Pierce Protease Inhibitor mini tablet
Pierce Protease Inhibitor tablets, EDTA-free
Pierce Protease Inhibitor mini tablets, EDTA-free
Pierce Protease Inhibitor XL Capsules, EDTA-free
Phosphatase liquid cocktail and tablets Halt Phosphatase Inhibitor Cocktail
Pierce Phosphatase Inhibitor Tablet
Combined protease and phosphatase liquid cocktails and tablets Halt Protease and Phosphatase Inhibitor Cocktail
Halt Protease and Phosphatase Inhibitor Cocktail, EDTA free
Pierce Protease and Phosphatase Inhibitor Mini Tablet
Pierce Protease and Phosphatase Inhibitor Mini Tablet, EDTA Free

Purify and enrich your protein sample

Various methods are used to enrich or purify a protein of interest from other proteins and components in a crude cell lysate or other sample. Ion exchange and affinity chromatography are two commonly used strategies for partial or 1-step purification.

Affinity purification

Also known as affinity chromatography, this purification method is enabled by the specific binding properties of a protein to an immobilized ligand. Since the protein of interest is tightly bound, contaminants can be removed through wash steps, and the bound protein can be stripped (eluted) from the support in a highly purified form. Affinity purification is desirable because it often produces higher protein yields and requires less steps than other purification methods. It is the method of choice for purifying recombinant or biotinylated proteins and antibodies.

Discover more about protein purification
See protein isolation and purification learning resource

Ion exchange (IEX) purification

Also known as ion exchange chromatography, this purification method enables the separation of proteins based on the protein charge at a particular pH. Since multiple proteins may have similar charges, IEX chromatography generally enables only partial purification of a protein of interest when used early in a multistep purification process. However, IEX resins can also be used during a final polishing step to remove specific contaminants that persist after other purification steps. Typically, proteins bind to the IEX column at low ionic strength and elute differentially by increasing salt concentration or changing pH in a gradient. A cation exchange resin binds to positively charged proteins; an anion exchange resin binds to negatively charged proteins. Ion exchange resins are classified as “weak” or “strong”, which refers to the extent that the ionization state of the functional groups varies with pH.

Discover more about ion exchange purification

How protein purification works

  1. Bind: Capturing protein of interest using a specific ligand.
  2. Wash: Remove unwanted proteins and other impurities, such as nucleic acids, during the wash phase.
  3. Elute: This step reverses the binding reaction. One can elute and enrich the target protein by varying buffering conditions such as pH, salt concentration, and detergents, or including competitive binders.
  4. Polishing: Using an additional chromatography step (usually ion-exchange or size exclusion) to remove remaining trace impurities and other contaminants by making best use of the known characteristics of the target protein.

Protein purification workflow

Chart of protein purity as a function of the number of purification steps in the purification method

Comparison of DYKDDDDK-tagged SUMO protein yield and background using Pierce anti-DYKDDDDK resin and other products C- and N-terminal DYKDDDDK-tagged SUMO proteins were expressed in E. coli and purified using Pierce Anti-DYKDDDDK Magnetic Agarose, Sigma-Aldrich® Anti-FLAG™ M2 Magnetic Beads, and MBL Anti-DDDDK-tag mAb-Magnetic Agarose. Tagged protein was competitively eluted with Pierce 3x DYKDDDDK Peptide, and the results were analyzed by SDS-PAGE (A) and densitometry using the Invitrogen iBright Imaging System (B). Comparison between the starting lysate and elution fractions shows effective immunoprecipitation and elution of DYKDDDDK-tagged protein, with minimal background from the Pierce magnetic agarose compared to the other suppliers’ products.

Protein purification product features:

  • Broad product selection—strong ion exchange and affinity supports for the purification and enrichment of proteins and antibodies; affinity ligands enable 1-step purification of recombinant and biotinylated proteins, while activated supports provide a platform for custom protein immobilization
  • High performance—resins are designed to maximize protein yield and reduce background
  • More formats—magnetic beads, loose resins, FPLC cartridges, and 96-well filter plates enable protein purification from screening and small-scale phases to process-scale purification
  • Economical—pricing that is similar to or better than other leading suppliers
Types of affinity purification Description
Protein immobilization
(Activated supports for custom immobilization)
Uses activated supports and accessories for the immobilization of proteins, antibodies, and other molecules. These resins or magnetic beads are available separately or in convenient kits. Different reactive chemistries are available to optimize immobilization based on the ligand properties.

See covalent immobilization of affinity ligands learning resource
Antibody purification Proteins A, G, A/G, and L have unique properties, which make each one suitable for different types of antibody targets (e.g., antibody subclass or animal species). These ligands enable purification of general immunoglobulins from a crude sample. Depending on the sample source, an antigen-specific antibody may account for only a small portion of the total immunoglobulin in the sample. For example, generally only 2–5% of total IgG in mouse serum is specific for the antigen used to immunize the animal.

See antibody purification learning resource
Recombinant protein purification
(Fusion protein purification)
Uses resins for the purification of recombinant proteins from cultures such as E. coli or Pichia. These resins are available in multiple formats to accommodate a variety of needs, from high-throughput screening to batch and pilot-scale purification. Superflow resins have undergone extensive chemical characterization. We have ligands targeting a variety of fusion tags, including 6xHis, GST, anti-DYKDDDDK (anti-FLAG™), c-Myc, and HA.

See fusion protein learning resource
Purification using magnetic Agarose Magnetic agarose beads consist of highly crosslinked agarose encapsulating a ferrimagnetic core. The beads are 10–40 uM in size and have higher binding capacity than traditional magnetic beads. We offer a variety of ligands for immunoprecipitation (IP), co-immunoprecipitation (co-IP), pull-down, and other high throughput affinity screening applications, utilizing immobilized Protein A/G, Ni-NTA, Glutathione, and Anti-DYKDDDDK. The beads are removed from the solution manually using a magnetic stand or by automation using an instrument such as the Thermo Scientific KingFisher Flex Magnetic Particle Processor. Automated instruments are especially useful for higher throughput purification and screening of purification conditions.

See scientific poster: High capacity magnetic supports for automated antibody and epitope-tagged protein purifications
Biotin affinity purification Uses resins for the purification of biotinylated or desthiobiotinylated proteins, peptides, and other molecules. These resins are available in multiple pack sizes, as well as in spin columns, kits, FPLC cartridges, and coated plates. Different biotin-binding ligands are available based on elution conditions or level of purity.

See avidin-biotin interaction learning resource
Immunoprecipitation (IP) Small-scale affinity purification of antigens using a specific antibody that is immobilized to a solid support such as magnetic beads or agarose resin. IP is one of the most widely used methods for isolation of proteins and other biomolecules from cell or tissue lysates for the purpose of subsequent detection by western blotting and other assay techniques.

See immunoprecipitation learning resource
See IP support
Co-immunoprecipitation (co-IP) and pull-down Similar to IP, except the target antigen precipitated by the antibody is used to co-precipitate its binding partner(s) or associated protein complex from the lysate and pull-downs. This method is used when antibodies to specific proteins are not available. These “bait” proteins are tagged with an epitope to which a high-affinity antibody is available and ectopically expressed in the cell of interest.

See co-immunoprecipitation learning resource
See pull-down assays learning resource
See co-IP and pull-down support
ChIP, RIP, and protein-nucleic acid pull-down Chromatin immunoprecipitation (ChIP) assays are performed to identify regions of the genome with which DNA-binding proteins, such as transcription factors and histones, associate. In ChIP assays, proteins bound to DNA are temporarily crosslinked and the DNA is sheared prior to cell lysis. The target proteins are immunoprecipitated along with the crosslinked nucleotide sequences, and the DNA is then removed and identified by PCR, sequenced, applied to microarrays, or analyzed in some other way.

RNA immunoprecipitation (RIP) uses an approach similar to ChIP, except that RNA-binding proteins are immunoprecipitated instead of DNA-binding proteins. Immunoprecipitated RNAs can then be identified by RT-PCR and cDNA sequencing.

See ChIP learning resource
See protein-nucleic acid interaction support
Endotoxin detection and removal kits Endotoxin is a type of pyrogen and is a component of the exterior cell wall of Gram-negative bacteria. It is an unwanted and toxic by-product of recombinant proteins purified from E coli. Therefore, its removal from protein samples is an important step for downstream applications.

See app note: Eliminate endotoxins from protein and antibody samples

Overview of ion exchange, affinity, and activated supports

Application Purity level Ligand and/or chemistry Base bead type Packaging options
Ion exchange purification Medium to high (aication-specific) Strong anion exchange POROS Loose resin
Strong cation exchange
Antibody purification High Protein A, protein G, protein A/G Agarose, magnetic beads, magnetic agarose, POROS Loose resins or beads, spin columns and kits, chromatography cartridges, 96-well spin plates
Protein L Agarose, magnetic beads
Melon Gel Agarose
Fusion protein purification High Ni-NTA, Ni-IDA, cobalt, glutathione Agarose, Superflow, magnetic beads, magnetic agarose Loose resins or beads, spin columns and kits, chromatography cartridges, 96-well spin plates
Anti-c-Myc, anti-HA, anti-FLAG Agarose, UltraLink magnetic beads Loose resins or beads, kits
Biotin affinity purification High Avidin, streptavidin, NeutrAvidin, monomeric avidin Agarose, magnetic beads Loose resins, spin columns and kits, chromatography cartridges, 96-well spin plates
Protein immobilization High Amine-reactive, sulfhydryl-reactive, carbonyl-reactive, carboxyl-reactive Agarose Loose resins or dry powder
Epoxy, tosyl-activated, carboxylic acid, amine Magnetic beads Loose beads

Select your resin based on purification scale and application

Scale High-throughput screening High-throughput batch Batch Pilot Process
Description Small scale, automation compatible Lab or bench scale Lab or bench scale Scale-up desired Production scale
Yield Microgram Milligram Milligram Gram Kilogram
Format Magnetic particle processor Magnetic particle processor, 96-well spin plate (agarose) Gravity flow, spin column (agarose), fast protein liquid chromatography (FPLC) at low flow rates FPLC at medium flow rates FPLC at high flow rates
Application High-throughput screening, interaction studies (IP, co-IP, pull-down), mutational analysis High-throughput screening, interaction studies (IP, co-IP, pull-down), mutational analysis requiring mg scale Functional assays, structural analysis Structural analysis, intermediate-scale production Bulk production
Recommended resin type Magnetic bead (1-2.8 µm)  
  Magnetic agarose (10-40 µm)  
  Agarose (45-165 µm)  
  Superflow (45-165 µm)  
  UltraLink resin (50-80 µm)  
  POROS resin (50 µm)

Tips

The process of protein purification varies depending on the downstream analyses to be performed. Certain steps may be repeated or omitted to achieve the desired result.

Purification products

Protein immobilization

Product selection guides Products
Protein immobilization Pierce NHS-Activated Agarose
AminoLink Coupling Resin
AminoLink Plus Coupling Resin
SulfoLink Coupling Resin
GlycoLink Coupling Resin
CarboxyLink Coupling
Additional activated supports and accessories Pierce NHS-Activated Agarose Spin Columns
AminoLink Plus Immobilization Kit
AminoLink Plus Micro Immobilization Kit
AminoLink Immobilization Kit
AminoLink Reductant
Pierce CDI-activated Agarose Resin
SulfoLink Immobilization Kit for Peptides
UltraLink Iodoacetyl Micro Peptide Coupling Kit
GlycoLink Immobilization Kit
GlycoLink Micro Immobilization Kit
CarboxyLink Immobilization Kit

Antibody purification

Product selection guides Products
Antibody purification Protein A Magnetic beads Pierce Protein A Magnetic Beads
Dynabeads Protein A Magnetic Beads
Resins Pierce Protein A Agarose
Pierce Protein A Plus Agarose
Pierce Recombinant Protein A Agarose
POROS MabCapture A Select
Spin columns and kits NAb Protein A Plus Spin Columns
NAb Protein A Plus Spin Kits
Buffers Pierce Protein A IgG Binding Buffer
Protein G Magnetic beads Pierce Protein G Magnetic Beads
Dynabeads Protein G Magnetic Beads
Resins Pierce Protein G Agarose
Pierce Protein G Plus Agarose
POROS MabCapture G Select
Spin columns and kits NAb Protein G Spin Columns
NAb Protein G Spin Kits
Pierce Recombinant Protein G
Buffer Pierce Protein G IgG Binding Buffer
Protein A/G Magnetic beads Pierce Protein A/G Magnetic Agarose Beads
Pierce Protein A/G Magnetic Beads
Resins Pierce Protein A/G Agarose
Pierce Protein A/G Plus Agarose
POROS MabCapture A/G Select
Spin columns and kits NAb Protein A/G Spin Columns
NAb Protein A/G Spin Kit
Pierce Recombinant Protein A/G
Buffer Pierce Protein A/G IgG Binding Buffer
Protein L Magnetic beads Pierce Protein L Magnetic Beads
Resins Pierce Protein L Agarose
Pierce Protein L Plus Agarose
Spin columns and kits NAb Protein L Spin Columns
NAb Protein L Spin Kit
Pierce Recombinant Protein L
Melon Gel Melon Gel IgG Spin Purification Kit
Melon Gel IgG Purification Kit
Melon Gel Monoclonal IgG Purification Kit
Monoclonal IgG Purification Kit
Melon Gel Spin Plate Kit for IgG Screening
Melon Gel Chromatography Cartridges (1 mL or 5 mL)
Melon Gel Regenerant
Ascites Conditioning Reagent for Melon Gel
Melon Gel Purification Buffer
Buffers and related products Pierce IgG Elution Buffer
Pierce Gentle Ag/Ab Binding Buffer, pH 8.0
Pierce Gentle Ag/Ab Elution Buffer, pH 6.6
Pierce Gentle Ag/Ab Binding and Elution Buffer Kit
Pierce Saturated Ammonium Sulfate Solution
Pierce Thiophilic Adsorption Kit
Pierce Mannan Binding Protein Agarose
Pierce IgM Purification Kit
Elution Buffer for Pierce IgM Purification Kit
MBP Column Preparation Buffer for Pierce IgM Purification Kit
Pierce Jacalin Agarose

Recombinant protein purification

Product selection guides Products
His-tagged (6xHis) protein purification Pierce Ni-NTA Magnetic Beads
Pierce Ni-NTA Magnetic Agarose Beads
HisPur Ni-NTA Agarose Resin        
HisPur Ni-NTA Superflow Resin
HisPur Cobalt Agarose Resin
HisPur Cobalt Superflow Resin
GST-tagged protein purification Pierce Glutathione Magnetic Agarose Beads
Pierce Glutathione Agarose Resin
Pierce Glutathione Superflow Resin
Other epitope-tagged protein purification Pierce Anti-DYKD4K (FLAG) Magnetic Agarose
Pierce Anti-DYKD4K (FLAG) Affinity Resin UltraLink
Pierce Anti-c-Myc Magnetic Beads
Pierce Anti-c-Myc Agarose (Superflow 6)
Pierce Anti-HA Magnetic Beads
Pierce Anti-HA Agarose
Purification using magnetic agarose Pierce Ni-NTA Magnetic Agarose Beads
Pierce Glutathione Magnetic Agarose Beads
Pierce Anti-DYKD4K (FLAG) Magnetic Agarose Beads

Biotin affinity purification

Product selection guides Products
Biotin affinity purification Magnetic beads Pierce Streptavidin Magnetic Beads
Resins Pierce Avidin Agarose
Pierce Streptavidin Agarose
Pierce Streptavidin Agarose HC
Pierce NeutrAvidin Agarose
Pierce NeutrAvidin Agarose HC
Pierce Monomeric Avidin Agarose
Related products for biotin-binding applications Invitrogen CaptAvidin Agarose (Sedimented Bead Suspension)
Pierce Streptavidin Agarose Columns
Pierce High Capacity Streptavidin Chromatography Cartridge
Pierce Monomeric Avidin Agarose Kit
Pierce Biotin
Pierce Iminobiotin Agarose

IP, co-IP, pull-down

Product selection guides Products
Immunoprecipitation (IP) Antibody-binding IP products Protein A, G, A/G Magnetic beads
  • Dynabeads Protein A
  • Dynabeads Protein G
  • Pierce Protein A/G
Kits
  • Dynabeads Protein A kit
  • Dynabeads Protein G kit
  • Pierce Protein A/G kit
  • Pierce Crosslink Kit (A/G)
Secondary antibodies (anti-mouse, anti-rabbit) Magnetic beads
  • Dynabeads M-280, Sheep-anti Mouse IgG
  • Dynabeads M-280, Sheep-anti Rabbit IgG
Surface-activated beads (epoxy) Kits
Biotin-binding IP products
  • Pierce Streptavidin Magnetic Beads
  • Dynabeads M-280 Streptavidin
  • Dynabeads M-270 Streptavidin
  • Dynabeads MyOne Streptavidin C1
  • Dynabeads MyOne Streptavidin T1
  • DynaMag-2 Magnet
Recombinant protein (fusion tag) IP products
  • HisPur Ni-NTA Magnetic Beads
  • Pierce Ni-NTA Magnetic
  • Agarose Beads
  • Dynabeads His-Tag Isolation and Pulldown
  • Pierce Glutathione Magnetic Agarose Beads
  • Pierce Anti-DYKDDDDK Magnetic Agarose
  • Pierce Anti-HA Magnetic Beads
  • Pierce HA-Tag Magnetic IP/Co-IP Kit
  • Pierce Anti-c-Myc Magnetic Beads
  • Pierce c-Myc-Tag Magnetic IP/Co-IP Kit
  • DynaMag-2 Magnet
Co-immunoprecipitation (co-IP) and pull-down Spin column
Magnetic beads
ChIP, RIP, and Protein-Nucleic Acid Pull-Down ChIP
  • MAGnify Chromatin Immunoprecipitation System
  • Pierce Magnetic ChIP Kit
  • Pierce ChIP-grade Protein A/G Magnetic Beads
  • Proteinase K Solution, ChIP grade
RIP
  • Pierce Magnetic RNA-Protein Pull-Down Kit
  • Pierce Streptavidin Magnetic Beads
  • Pierce RNA 3' End Desthiobiotinylation Kit

Ion exchange purification

Product selection guides Products
Ion exchange purification Ion exchange spin columns Pierce (SCX) Spin Columns
Pierce (SAX) Spin Columns
Ion Exchange Purification
Ion exchange resins POROS XS Resin
POROS XQ Resin
POROS 50 HQ Resin

Endotoxin removal

Product selection guides Products
Endotoxin detection Pierce Chromogenic Endotoxin Quant Kit
Pierce LAL Chromogenic Endotoxin Quantitation Kit
Endotoxin removal Loose resin
Spin columns (0.25 mL, 0.5 mL, or 1 mL)

Clean up your protein sample

Many detergents and salts used in protein extraction formulations may have adverse effects on protein function or stability, or may interfere with downstream analysis. Therefore, it may be necessary to remove or reduce these contaminants following cell lysis or subsequent sample processing such as protein purification.

Download Protein Clean-Up Technical Handbook

Discover more about protein dialysis, desalting, and concentration

Tips

  • If the protein concentration is too dilute for further processing or analysis, the sample can be concentrated quickly using centrifugal concentrators.
  • For buffer exchange during concentration technique, the retentate can be diluted with exchange buffer and centrifuged. This process can be repeated until the desired level of exchange or desalting has been achieved.

Protein clean-up products

Product category Products
Protein dialysis Slide-A-Lyzer products
Protein desalting Zeba products
Protein concentration Protein concentrators
Detergent removal products HiPPR Detergent Removal 96-well Spin Plates
Abundant protein depletion High Select Top 14 and Top 2 reagents
Small molecule removal Biotin, dye, crosslinkers, and reducing agent removal products

Protein dialysis

Dialysis is a classic clean-up technique that removes small molecules and unwanted compounds by selective diffusion through a semi-permeable membrane. A sample and a buffer solution are placed on opposite sides of the membrane. Proteins that are larger than the membrane pores are retained on the sample side of the membrane, but smaller molecules (contaminants) diffuse freely through the membrane until an equilibrium concentration is achieved. Through this technique, the concentration of small contaminants in the sample can be decreased to acceptable levels.

Protein dialysis products features:

  • Excellent sample recovery—low-binding plastics and membranes help minimize sample loss compared to filtration and resin systems
  • Convenience—easy-to-grip design helps simplify sample addition and removal with syringe and/or pipette
  • Secure—sealed membranes help prevent leakage that can occur with dialysis tubing and homemade devices
  • Validated—each device is be leak-tested during production

Discover more about protein dialysis
See protein dialysis learning resource

Product highlight

MWCO* membrane 10-100 µL
Pierce 96-well Microanalysis Plate
10-2,000 µL
Slide-A-Lyzer MINI Dialysis Device
0.1-70 mL
Slide-A-Lyzer G2 Dialysis Cassette
0.1-30mL
Slide-A-Lyzer Dialysis Cassette
150-250 mL
Slide-A-Lyzer Dialysis Flask
15-100 mL
SnakeSkin Dialysis Tubing
2K N/A
See product
N/A
3.5K
See product

See product
7K N/A N/A
10K
See product

See product
20K N/A
See product
N/A
See full comparison of these products ›

*MWCO: Molecular weight cut off.

Protein recovery by molecular weight cutoff (MWCO)

Sample retention by the 2K, 3.5K, 7K, 10K, and 20K MWCO Thermo Scientific Slide-A-Lyzer cassette membrane. Individual proteins or vitamin B12 (1 mg/mL) in either saline or 0.2 M carbonate-bicarbonate buffer, pH 9.4 were dialyzed overnight (17 hours) at 4°C. The amount of retentate was estimated using either the Pierce BCA Protein Assay Kit or absorption at 360 nm (for vitamin B12).

Dialysis rates for various formats

The rate of removal of NaCl using various dialysis products. NaCl removal from samples was determined by measuring the conductivity of the retentate at the indicated times. (A) Slide-A-Lyzer MINI Dialysis Device (10K MWCO, 2 mL) versus conventional dialysis. Bovine serum albumin (BSA) samples (2 mL, 0.25 mg/mL in 1 M NaCl) were dialyzed against 45 mL of water in 50 mL disposable conical tubes on an orbital shaker (300 rpm) at room temperature. The water was changed once after 2 hours. Results are the average of two samples. For conventional dialysis, the samples were dialyzed against 2 L of water in a beaker with stirring. Greater than 95% of NaCl was removed within 4 hours. (B) Samples of 0.1 mL (0.4 mg/mL cytochrome C containing 1 M NaCl) were dialyzed in the Pierce 96-well Microdialysis Plate against 1.8 mL of water at RT with gentle shaking. The buffer was changed at 1-, 2-, and 3-hour intervals over a 4-hour period. Removal of NaCl was >83% after 2 hours and >99% after 4 hours. (C) Proteins in 200 mL samples containing 1 M NaCl were dialyzed at room temperature using Slide-A-Lyzer Dialysis Flasks with 2K, 3.5K, 10K, and 20K MWCOs. The dialysis buffer (4 L) was changed after 2 and 5 hours (triangles; also at 41 hours for the 2K condition). Greater than 95% of NaCl was removed within 8 to 18 hours (41 hours for the 2K condition).

Protein desalting

Size exclusion chromatography, also described as gel filtration, can be used for removal of salts from samples. Using this technique, a resin is selected with pores large enough for salts to penetrate, but small for the protein of interest to enter. This causes contaminants to slow down their rate of migration. The larger and faster proteins separate from the slower and smaller molecules during gravity flow or centrifugation.

Discover more about protein desalting
See protein desalting learning resource

Protein desalting products features:

  • High performance—proprietary resin enables excellent protein recovery and efficient contaminant removal
  • Flexible—available in spin columns, filter spin plates, and cartridges for a range of needs
  • Fast—no fraction screening or waiting for protein to emerge by gravity flow
  • Economical—cost-effective products that offer great performance

Product highlight

Type Spin columns Spin plates Chromatography columns
Format Micro 0.5 mL 2 mL 5 mL 10 mL 96-well 1 mL 5 mL
Resin bed 75 µL 0.5 mL 2 mL 5 mL 10 mL 550 µL 1 mL 5 mL
Sample volume (7K MWCO) 2-12 µL 30-130 µL 200-700 µL 500-2,000 µL 700-4,000 µL 20-100 µL 50-250 µL 100-1,500 µL
Sample volume (40K MWCO) 5-14 µL 70-200 µL 200-900 µL 300-2,000 µL 1,000-4,000 µL 20-100 µL N/A N/A
See full comparison of these products ›

Comparison of protein recovery and sample dilution

Zeba Spin Desalting Columns result in a high protein recovery while providing minimal sample dilution over a wider range of sample concentrations and volumes compared to alternative products. Zeba Spin Desalting Columns, 10 mL (7K MWCO) and GE PD-10 Columns were used to desalt 1.5, 2.5, and 3.5 mL BSA samples at a concentration of 0.04, 0.2, and 1 mg/mL. Desalting was performed according to the manufacturers’ recommended protocols; both the spin and gravity protocols were used for the GE PD-10. Protein recovery was analyzed by SDS-PAGE. For each electrophoresis gel, an aliquot of starting sample equal to 1 μg of BSA was loaded in lane 1 as the loading control; all other desalted samples were loaded in the gel at the same volume as the loading control. Differences in intensity between lanes are a combination of protein recovery and sample dilution caused by desalting. The largest differences in recovery and concentration are noted in the highlighted area.

Protein concentrators

Protein concentration is similar to dialysis and uses a semi-permeable membrane to separate proteins from low molecular weight compounds. Unlike dialysis, which relies on passive diffusion, concentration is achieved by forcing solution through membrane using centrifugation. During centrifugation, both buffer and low molecular weight solutes are forced through the membrane where they are collected on the other side (filtrate). Macromolecules (proteins) remain on the sample side of the membrane, where they become concentrated to a smaller volume (retentate), as the reagent is forced across the membrane to the other side.

Discover more about protein concentrators
See protein dialysis, desalting, and buffer exchange learning resource

Protein concentrator product features

  • Rapid processing—unique design minimizes membrane fouling; and sample concentration of 10- to 30-fold can be achieved in 5–30 minutes for 10K MWCO (device-dependent times may vary for other MWCOs), even with particle-laden solutions
  • High recovery—retain >90% of protein samples while removing contaminants or exchanging buffers
  • Convenient—clear markings, wide sample chamber, and removable filtrate chamber make handling simple and easy
  • Instrument compatible—can be used with standard centrifuges utilizing either fixed-angle or swinging-bucket rotors

Product highlight

Volume range 0.1–0.5 mL 2–6 mL 5–20 mL 20–100 mL
MWCOs available 3K, 10K, 30K, 100K 3K, 10K, 30K, 100K 3K, 10K, 30K, 100K 5K, 10K, 30K, 100K
Processing time* 3-15 min 15-90 min 15-60 min 15-90 min
Retentate volume range* 9-67 µL 51-174 µL 121-777 µL 1.9-3.5 mL
Protein recovery range* 95-100% 94-100% 94-100% 92-98%
See full comparison of these products ›

*Four different protein solutions were used for each MWCO

Comparison of protein recovery between Pierce Protein Concentrators (using 3K, 5K, 10K, 30K, or 100K MWCO) and other vendors for 0.5 mL, 6 mL, 20 mL, and 100 mL concentrators. Samples of different protein solutions were centrifuged in Pierce Protein Concentrators and other suppliers’ concentrators according to manufacturers’ instructions: 0.5 mL (15,000 x g), 5 mL (4,000 x g), 20 mL (4,700 x g), and 100 mL (1,200 x g). Samples were centrifuged until a greater than 15- to 30-fold decrease in sample volume was achieved; protein concentration was measured by either Pierce BCA Protein Assay Kit (0.5 mL concentrators only) or absorbance at A280.

Quantify and detect your target protein

There are various methods that can be used to detect and measure your target protein depending on your experimental needs. Below are common techniques used to detect and measure proteins from complex mixtures (e.g., lysates, sera) and the typical requirements for each method.

Total protein quantitation

Quantifying total protein concentration is an important step in workflows involving isolation, separation, and analysis of proteins by biochemical methods. Assay methods may use colorimetric or fluorescent detection with fluorometers, spectrophotometers, or plate readers. Every protein assay has limitations depending on the application and the specific protein sample analyzed. The most useful features to consider when choosing a protein assay are sensitivity (lower detection limit), compatibility with common substances in samples (e.g., detergents, reducing agents, chaotropic agents, inhibitors, salts, and buffers), standard curve linearity, and protein-to-protein variation.

Colorimetric protein assays

Colorimetric signals can be detected using a microplate reader or spectrophotometer. The most popular colorimetric protein assays are:

  • BCA Assays—Protein-copper chelation with secondary detection of the reduced copper.
  • Bradford Assays—Protein-dye binding with direct detection of the color change associated with the bound dye.
     

Fluorescent protein assays

Fluorescence-based protein quantitation is an alternative to colorimetric methods. Fluorescence detection methods offer excellent sensitivity, requiring less protein sample thereby leaving more sample available for your experiment. Additionally, read time is not a critical factor, so the assays can be readily adapted for automated high-throughput applications. The fluorescence signal can be detected using a fluorometer or microplate reader.

Discover more about protein assays and analysis
See protein quantitation and detection learning resource
Learn more about methods and technologies for identifying and measuring proteins

Product highlight Category Description
Pierce BCA Protein Assay Kit Colorimetric Two-component, high-precision, detergent-compatible protein assay. Compared to most dye-binding methods, the BCA assay is affected much less by protein compositional differences, providing greater concentration accuracy.
Pierce Rapid Gold BCA Protein Assay Kit Colorimetric This kit maintains the key characteristics of the traditional BCA assay but allows a fast time and room temperature incubation equal to dye-binding methods.
Quant-iT Protein Assay Fluorescence The assay is highly selective for protein and exhibits very little protein-to-protein variation. The assay is performed at room temperature, and the signal is stable for 3 hours.
Qubit 4 Fluorometer Fluorometer The Qubit 4 Fluorometer is the latest version of the popular Qubit fluorometer designed to accurately measure protein quantity.

Use the interactive Protein Assay Selection Guide

Tips

  • No one reagent is the ideal or best protein assay method. Each method has its advantages and disadvantages.
    Use the interactive Protein Assay Selection Guide to filter products based on sample type, assay time, read-out (colorimetric or fluorescent), and compatibility to detergents or reducing agents
  • To learn about the different ways to quantitate protein concentrations continue reading the article: Overview of protein assays.

Protein detection

Protein detection method attributes ELISA Western blotting Mass spec
Advantages
  • High-throughput capability with 96-well or 384-well plate formats
  • Quantification of target proteins
  • Identification and verification of molecular weight
  • Ability to separate and isolate protein of interest
  • Identification and quantitation of multiple targets from the same sample
  • Detection of post-translational modifications or different isotypes
Sensitivity <5-10 pg/mL low femtogram to high attogram* attomolar range (1018)
Lysis buffer compatibility
  • For non-activity based ELISAs: ionic detergent based lysis buffers
  • For activity based ELISAs: non-ionic detergent-based lysis buffers (e.g. NP-40, Triton X-100)
  • For SDS-PAGE (denaturing): RIPA or other lysis buffers with ionic detergents
  • For native-PAGE applications: non-ionic detergents-based lysis buffers (e.g. NP-40, Triton X-100)
Detergents and high salts must be removed prior to analysis
Typical total protein required 0.1 µg/mL - 1 µ g/mL 1 µg- 50 µg <1 µg
Equipment required Plate reader X-ray film or CCD imaging equipment Mass spectrometer

*With high sensitivity HRP substrates, such as SuperSignal West Atto Ultimate Sensitivity Substrate

Protein quantitation and detection products


References

Resources

Support

  • Support Centers
    Relevant technical information, tips and tricks, and answers to everyday problems
  • Contact us
    Get assistance from one of our experts